One of the goals of plant genetic engineering is to produce plants with agronomically desirable characteristics or traits. The proper expression of a desirable transgene in a transgenic plant is one way to achieve this goal. Elements having gene regulatory activity, i.e. regulatory elements such as promoters, leaders, introns and transcription termination regions, are non-coding polynucleotide molecules which play an integral part in the overall expression of genes in living cells. Isolated regulatory elements that function in plants are therefore useful for modifying plant phenotypes through the methods of genetic engineering.
Many regulatory elements are available and are useful for providing good overall gene expression. For example, constitutive promoters such as P-FMV, the promoter from the 35S transcript of the Figwort mosaic virus (U.S. Pat. No. 6,051,753); P-CaMV 35S, the promoter from the 35S RNA transcript of the Cauliflower mosaic virus (U.S. Pat. No. 5,530,196); P-Corn Actin 1, the promoter from the actin 1 gene of Oryza sativa (U.S. Pat. No. 5,641,876); and P-NOS, the promoter from the nopaline synthase gene of Agrobacterium tumefaciens are known to provide some level of gene expression in most or all of the tissues of a plant during most or all of the plant's lifespan. While previous work has provided a number of regulatory elements useful to affect gene expression in transgenic plants, there is still a great need for novel regulatory elements with beneficial expression characteristics. Many previously identified regulatory elements fail to provide the patterns or levels of expression required to fully realize the benefits of expression of selected genes in transgenic crop plants. One example of this is the need for regulatory elements capable of driving gene expression in different types of tissues.
Promoters
The genetic enhancement of plants and seeds provides significant benefits to society. For example, plants and seeds may be enhanced to have desirable agricultural, biosynthetic, commercial, chemical, insecticidal, industrial, nutritional, or pharmaceutical properties. Despite the availability of many molecular tools, however, the genetic modification of plants and seeds is often constrained by an insufficient or poorly localized expression of the engineered transgene.
Many intracellular processes may impact overall transgene expression, including transcription, translation, protein assembly and folding, methylation, phosphorylation, transport, and proteolysis. Intervention in one or more of these processes can increase the amount of transgene expression in genetically engineered plants and seeds. For example, raising the steady-state level of mRNA in the cytosol often yields an increased accumulation of transgene expression. Many factors may contribute to increasing the steady-state level of an mRNA in the cytosol, including the rate of transcription, promoter strength and other regulatory features of the promoter, efficiency of mRNA processing, and the overall stability of the mRNA.
Among these factors, the promoter plays a central role. Along the promoter, the transcription machinery is assembled and transcription is initiated. This early step is often rate-limiting relative to subsequent stages of protein production. Transcription initiation at the promoter may be regulated in several ways. For example, a promoter may be induced by the presence of a particular compound or external stimuli, express a gene only in a specific tissue, express a gene during a specific stage of development, or constitutively express a gene. Thus, transcription of a transgene may be regulated by operably linking the coding sequence to promoters with different regulatory characteristics. Accordingly, regulatory elements such as promoters, play a pivotal role in enhancing the agronomic, pharmaceutical or nutritional value of crops.
At least two types of information are useful in predicting promoter regions within a genomic DNA sequence. First, promoters may be identified on the basis of their sequence “content,” such as transcription factor binding sites and various known promoter motifs. (Stormo, Genome Research 10: 394-397 (2000)). Such signals may be identified by computer programs that identify sites associated with promoters, such as TATA boxes and transcription factor (TF) binding sites.
Second, promoters may be identified on the basis of their “location,” i.e. their proximity to a known or suspected coding sequence. (Stormo, Genome Research 10: 394-397 (2000)). Promoters are typically contained within a region of DNA extending approximately 150-1500 basepairs in the 5′ direction from the start codon of a coding sequence. Thus, promoter regions may be identified by locating the start codon of a coding sequence, and moving beyond the start codon in the 5′ direction to locate the promoter region.
Corn
Maize (Zea mays L.), or corn, is one of the three most important cereal crops in the world. Maize is high yielding, easy to process, readily digested, and costs less than other cereals. It is also a versatile crop, allowing it to grow across a range of agroecological zones. Every part of the maize plant has economic value: the grain, leaves, stalk, tassel, and cob can all be used to produce a large variety of food and non-food products. Zea mays Linnaeus, known as maize throughout most of the world, and as corn in the United States, is a large, annual, monoecious grass, that is grown for animal feed, silage, human grain, vegetable oil, sugar syrups, and other miscellaneous uses. It is the premier cash crop in the United States, and its cultivation, genetics, processing, financing, and distribution on a national and international scale is pervasive and complex.
Maize is used primarily as a staple food for human consumption, animal feed. and as raw material for industrial use. It is also used as seed. In industrialized countries, a larger proportion of maize is used for livestock feeding and as industrial raw material for food and non-food uses. On the other hand, the bulk of maize produced in developing countries is used as human food although its use as animal feed is increasing. An understanding of trends in maize processing/utilization is of timely interest and importance.
According to FAO data, 589 million metric tons of maize were produced worldwide in 2000, on 138 million hectares. The United States was the largest maize producer (43% of world production) followed by Asia (25%) and Latin America and the Caribbean (13%). Africa produced 7% of the world's maize. The world average yield in 2000 was 4255 kg per hectare. Average yield in the USA was 8600 kg per hectare, while in sub-Saharan Africa it was 1316 kg per hectare. Corn has the highest value of production of any United States crop: its 1987 value was 12.1 billion dollars, compared to soybeans at 10.4, hay at 9.1, wheat at 5.4, and cotton at 5.0.
Corn has been cultivated since the earliest historic times from Peru to central North America. The region of origin is now presumed to be Mexico (Gould, 1968). Dispersal to the Old World is generally deemed to have occurred in the sixteenth and seventeenth centuries (Cobley and Steele, 1976); however, recent evidence indicates that dispersal to India may have occurred prior to the twelfth and thirteenth centuries by unknown means (Johannessen and Parker, 1989).
In industrialized countries maize is largely used as livestock feed and as a raw material for industrial products, while in low-income countries it is mainly used for human consumption. In sub-Saharan Africa, maize is a staple food for an estimated 50% of the population. It is an important source of carbohydrate, protein, iron, vitamin B, and minerals.
Throughout the tropics and subtropics most maize is grown by small-scale farmers, generally for subsistence as part of agricultural systems that feature several crops and sometimes livestock production. The systems often lack inputs such as fertilizer, improved seed, irrigation, and labor. Most maize-producing countries in the industrialized world employ intensive input and highly mechanized monocropping production systems. Hybrid maize varieties are commonly used.
Threats to the maize plant include pests, weeds and drought. Major insect pests include stem and ear borers, armyworms, cutworms, rootworm, grain moths, beetles (weevils, grain borers, rootworms, and whitegrubs), and virus vectors (aphids and leafhoppers). A range of pathogens, primarily fungi, also damage the maize plant. Weeds often cause severe maize yield losses because they compete for nutrients, light, and moisture. In the Nigerian savanna, for example, weed-related yield losses ranging from 65% to 92% have been recorded. Last but not least, periodic drought caused by irregular rainfall distribution reduces maize yields by an average of 15% each year, which is equivalent to at least US$ 200 million in foregone grain. The effects of prolonged droughts, such as those that have struck Eastern and Southern Africa in recent years, have been disastrous.
For these reasons and others, it is of immense social, ecological and economic interests to develop corn plants that have enhanced nutrition, improved resistance to pests, and tolerance to harsh conditions such as drought. Thus, the identification of new genes, promoters and other regulatory elements. that function in corn is useful not only in developing enhanced varieties of maize, but also in developing enhanced varieties of other crops. In particular, developments in corn are applicable to other cereal crops, such as sorghum, corn, barley and wheat. Clearly, there exists a need in the art for new regulatory elements, such as promoters, that are capable of expressing heterologous nucleic acid sequences in important crop species.